A low molecular weight, reversible, species-independent suppressor of normal and malignant lymphocyte division (LMWS), is released by metabolizing rat spleen cells into their culture supernatant. Large volumes of this supernatant have been produced and the active product isolated by ultrafiltration and Sephadex G-10 chromatography. Partial purification of this batch preparation will be achieved by chromatography on cation and anion exchange resins. Final purification will be performed by high pressure liquid chromatography, using appropriate carbon-silica bonded ion exchange resins. The purity of biologically active material and the number of chemical components will be determined by ultraviolet and nuclear magnetic resonance spectroscopy, and by thin layer chromatography using several solvent systems. As single component purity is approached, the active molecule will be subjected to structural analysis by nuclear magnetic resonance and mass spectroscopy, and X-ray diffraction crystallography. The biological effects of each fraction on unstimulated and stimulated, normal and malignant lymphoid targets will be assessed in vitro, using radiolabel and mithramycin assays to evaluate DNA synthesis, and a mitotic index assay to directly measure cell growth. The more highly purified fractions of LMWS will be tested for their in vivo effects on the recurrent growth of murine lymphoid ascites tumors and the initial growth of lymphoblastic leukemias in order to evaluate their potential usefulness in the chemotherapy of lymphoid malignancies. In vivo studies of the excretion, toxicity and therapeutic index will be performed in the murine system. Highly purified fractions of LMWS will be screened for their effects on a panel of malignant human lymphoid cells from patients with leukemia and lymphoma, in final preparation for their testing in subhuman primates and human subjects.